Programmed cell death, or apoptosis, plays crucial roles in both development and tissue homeostasis. Apoptosis is widely observed in the nervous system during development. However, the molecular mechanisms underlying why only certain cells are selected to die and the execution of neural cell death itself are largely unknown. Recent genetic studies in mice and Drosophila revealed crucial roles for JNK (c-jun N-terminal kinase) activation in neural cell death. The JNK-mediated initiation of apoptotic mechanisms appears to be an excellent strategy for the fine-tuning of morphogenesis as well as cell selection in nervous tissue.

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OH radicals play a major role in radiation-induced DNA and cell membrane damage. These types of damage can also induce death by apoptosis through activation of a pro-apoptosis pathway. We attempted to detect OH radicals inside human promyelocytic leukemia (HL60) cells and estimate the relationship between radiation-induced apoptosis and OH radicals generated inside the cells. Electron spin resonance spectroscopy showed that OH radicals were generated by X-rays within irradiated cell pellets and the relative signal intensities of OH radicals increased with the radiation dose. Agarose gel electrophoresis revealed that the death of HL60 cells by apoptosis was accompanied by internucleosomal DNA fragmentation at 2 h after irradiation with 10-30 Gy. On ultrastructure evaluation by transmission electron microscopy, certain irradiated HL60 cells demonstrated condensed chromatin forms at the nuclear membrane and nuclear fragmentation. The frequency of apoptotic cells with condensation and fragmentation of nuclear chromatin increased with radiation dose in semithin sections. The increase of quantitative DNA fragmentation and percentage of non-living cells also correlated with radiation dose. These results suggest that OH radicals are generated inside cells before apoptosis occurs. The amount of OH radicals generated correlates with apoptotic cell death.

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The cortical peritubular interstitium of the normal kidney contains both fibroblasts and antigen-presenting dendritic cells. Characteristics of these interstitial cells were analyzed in an overnutrition model by electron microscopy after the cold-dehydration technique and immunohistochemistry for antigen-presenting cells. In control rats, fibroblasts and dendritic cells were clearly identified by electron microscopy on the basis of their distinct ultrastructures. Fibroblasts possessed slender cell processes, and contained an abundance of actin filament bundles occasionally anchoring to surrounding structures, whereas dendritic cells possessed irregularly-shaped cell processes with a clear cytoplasm and a paucity of actin filament bundles. In the experimental kidney from diabetic rats given a high cholesterol diet, the peritubular interstitium contained fibroblasts and vacuolated cells, and the extracellular matrices such as collagen bundles were distinctly increased compared with the control rat kidney. Immununohistochemical staining with OX6 and ED1 revealed that the peritubular interstitium in the control rat kidney contained dendritic cells, while that in the experimental rats was occupied by macrophages. The present study provides the first evidence indicating that overnutrition may dramatically affect the immune cells in nonlymphoid tissue.

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This study investigated the anatomical and neuropeptidergic properties of the duodenal neurons projecting to the gallbladder in the golden hamster. Fast blue (FB) was injected into the subserosa of the gallbladder in order to identify by retrograde tracing the duodenal neurons that project to the gallbladder. Subsequently, immunofluorescence microscopy was employed to see whether these duodenal neurons contained putative peptidergic neurotransmitters such as calcitonin gene-related peptide (CGRP), galanin (GAL) and vasoactive intestinal polypeptide (VIP).
  The FB-labeled cells were only found in the duodenal region adjacent to the major duodenal papilla where the biliary duct opens. On the other hand, there was no difference within this duodenal region in the numbers of FB-labeled cells between the mesenteric and anti-mesenteric portions, suggesting that these two portions of the duodenum equally contribute neuronal projections to the gallbladder. Double-immunofluorescence microscopy clearly demonstrated that a small population of FB-positive duodenal neurons contained putative neurotransmitters CGRP, GAL and VIP.
  Our data suggest that duodenal neurons around the major duodenal papilla in the golden hamster project to the gallbladder and exert their influence on the gallbladder via neuropeptides such as CGRP, GAL and VIP.

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Morphological studies examined communication routes between the epithelium and lamina propria mucosae in the human esophagus, using a series of techniques including silver staining, immunohistochemistry, transmission electron microscopy, and scanning electron microscopy (SEM). For SEM, tissue blocks were treated with either osmium/ultrasonication or NaOH. Observations showed the esophageal papillae to be arranged regularly in a mostly longitudinal row. The reticular fibers, consisting of fibrils approximately 40 nm in diameter, were situated just beneath the epithelial basal lamina. They showed a positive reaction with a type III collagen antibody, and formed a continuous sheet 2-3 μm thick with dense networks. This sheet as well as the epithelial basal lamina had numerous foramina of diameters of 3-5 μm. Immune cells such as lymphocytes and Langerhans cells were situated around these foramina. The foramina were situated both around papillae and the duct orifice of the esophageal gland. In addition, Iymphoid follicles surrounded the duct of the esophageal gland. The structural characteristics around the duct appear to be those of duct-associated lymphoid tissue (DALT). Thus, these foramina in the epithelial basal lamina and reticular fiber sheet may represent important communication routes between the epithelium and lamina propria mucosae. In addition, they may play an important role in the mucosal immune response in the human esophagus.

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We recently reported the presence of high concentrations of a Ca-binding matrix in the circumpulpal dentin of rat incisors which had been prevented from mineralization by a systemic administration of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP), a type of bisphosphonates, thus suggesting the role of the putative Ca-binding matrix in the appositional mineralization of circumpulpal dentin (TAKANO et al., 1998, 2000; OHMA et al., 2000). In this study, we examined the distribution of Ca-binding domains in the pulp tissue of normal rat teeth and its changes under the influence of HEBP, in order to identify and clarify the role of the Ca-binding matrix in the physiological process of dentin mineralization.
  Observation of the normal rat tooth pulp showed occasional, tiny extracellular deposits of Ca-enriched material in the odontoblast layer, associated primarily with pericapillary regions. Such deposits were immunopositive for dentin sialoprotein (DSP), displayed high levels of X-ray peaks for calcium and phosphorus, and showed a drastic increase in amount by daily injections of HEBP. A brief vascular perfusion of high Ca-containing solution in normal animals caused the extensive deposition of Ca-P complexes along the basolateral membranes of odontoblasts but not in the other regions of the pulp tissue.
  These data suggest the existence of DSP-enriched extracellular Ca-binding domains in the odontoblast layer and also indicate a novel Ca-binding property of the basolateral membranes of odontoblasts. Since DSP is primarily synthesized as dentin sialophosphoprotein (DSPP) and later cleaved into dentin phosphophoryn (DPP) and DSP in odontoblasts, and since DSP has no notable affinity for Ca, the sites of DSP-immunopositive Ca-P deposits in the odontoblast layer may also contain DPP, a highly phosphorylated acidic protein having a strong binding property for calcium. Characteristic Ca-binding properties seen in the odontoblast layer appear to be related to the regulation of the appositional mineralization of circumpulpal dentin.

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To demonstrate the structure of angiogenic blood vessels three-dimensionally, a gelatin sponge sheet immersed in a vascular endothelial growth factor (VEGF) solution was implanted in the rat dorsal muscular layer, and examined by light microscopy and scanning electron microscopy (SEM) 5 days to 2 weeks after implantation. Light microscopy of anti-collagen IV antibody immunostained specimens enabled a determination of the basement membrane tube of newly formed blood vessels in the implanted sponge sheet. The tubes were 5-40 μm in diameter, and sometimes tapered to a slender cord within the vascular network. The SEM study of 30% KOH treated tissues revealed two types of tapering ends of newly formed blood vessels. One consisted of endothelial cells with microprojections, and lacked any investment of pericytes over the length of 5-20 μm. The other type was a tapering tip of the endothelial tube covered with pericytic processes. The presence of long processes of pericytes extending beyond the tip of the endothelial tube and connecting to the adjacent vessel wall indicates that this type was produced by endothelial tube regression. Thus, the present study supports the ideas that endothelial tube formation is followed by pericyte coverage at the sprouting tip, and that endothelial tube regression precedes pericyte detachment at the regressing site.

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In a previous in situ hybridization study, we demonstrated the mRNA expression of napsin, an aspartic protease of the pepsin family, in the kidney, lung, and lymphoid organs of mice. However, findings on the cellular localization of napsin at the protein level are controversial, and no information on the subcellular localization is available. The present immunohistochemical study revealed the cellular and subcellular localization of napsin in mice and rats, and also analyzed the influences of chemical-induced proteinuria on the renal expression of this enzyme in rats. Immunohistochemistry using a polyclonal antibody against mouse napsin showed that napsin immunoreactivity was noticeable in lysosomes of renal proximal tubule cells and in lamellar bodies of pulmonary type II alveolar cells. In the lung, immunoreactivity was also found in lysosomes of alveolar macrophages and on the surface of type I alveolar cells; the immunoreactivities in these cells may be due to the uptake and adhesion of napsin secreted from type II alveolar cells, since they did not express napsin mRNA. Conversely, immunoreactivity for napsin was undetectable in B lymphocytes with intense mRNA expression. In puromycin- or doxorubicin-induced proteinuria, napsin mRNA expression was markedly elevated in renal proximal tubules, showing characteristic distribution patterns. Immunostaining of kidneys with proteinuria showed intense immunoreactivity for napsin in congested and enlarged lysosomes, called protein absorption droplets. These results indicate that napsin functions as a lysosomal protease and is involved in protein catabolism in renal proximal tubules.

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